title: Efficient chromatographic separation of intact proteins derivatized with a fluorogenic reagent for proteomics analysis.
abstract: To achieve more efficient separation of intact proteins for proteomics applications, three columns of differing diameters (4.0, 4.6 and 6.0 mm internal diameter) were chosen for comparison and investigated to identify optimal conditions. The column with the largest diameter gave the largest peak capacity, showing the efficient separation of intact proteins, such as two protein standards, glutathione S-transferase and β-lactoglobulin. On the other hand, a low-molecular-weight compound was separated effectively on the smaller diameter column, demonstrating that the separation mechanism seems to differ between high- and low-molecular-weight compounds. Finally, using the 6.0 mm i.d. column, 680 protein peaks were observed in mouse liver extracts, demonstrating that a wider diameter separation column is effective for intact protein separations.
authors.affiliation: Research Institute of Pharmaceutical Sciences, Musashino University, Tokyo, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Ichibangase
authors.forename: Itaru
authors.initials: I
authors.lastname: Yazawa
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Imai
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D008099:Liver / Q000737:chemistry; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D011506:Proteins / Q000032:analysis / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*
chemical_list: D011506:Proteins
keywords: HPLC separation; fluorogenic derivatization; intact proteins; peak capacity; proteomics